Protective activity of the lectin-like domain of TNF in permeability edema

TNF 凝集素样结构域对通透性水肿的保护活性

• 批准号: 7988150
• 负责人: Rudolf Lucas
• 金额: $ 37.25万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7988150
• 关键词: 5 year old; Affect; Affinity Chromatography; Agonist; Alveolar; Antibiotic Therapy; Arginine; Bacterial Infections; Bacterial Pneumonia; Bacterial exotoxin; Cause of Death; Cells; Cessation of life; Child; Cholesterol; Complication; Data; Development; Disease; Down-Regulation; Edema; Elderly; Enzyme Uncoupling; Enzymes; Epithelial; Epithelial Cells; Event; Exotoxins; Functional disorder; GTP-Binding Proteins; Generations; Goals; Human; In Vitro; Infection; Influenza A virus; Knock-in Mouse; Lead; Lectin; Life; Liquid substance; Listeria monocytogenes; Listeria monocytogenes hlyA protein; Lung; Mediating; Membrane; Mus; NADPH Oxidase; Patients; Peptide N-glycohydrolase F; Peptides; Permeability; Phosphorylation; Play; Pneumonia; Production; Protein Kinase C; Research; Role; Secondary to; Small Interfering RNA; Sodium; Sodium Channel; Sterility; Streptococcus pneumoniae; Streptococcus pneumoniae plY protein; TNF gene; Testing; Tissues; Toxin; Vascular Permeabilities; Virulence Factors; Virus Diseases; Wild Type Mouse; aged; arginase; base; combat; epithelial Na+ channel; improved; in vivo; inhibitor/antagonist; mortality; mouse model; mutant; novel therapeutic intervention; novel therapeutics; patch clamp; programs; protein expression; public health relevance; uptake

DESCRIPTION (provided by applicant): Severe pneumonia is the leading single cause of mortality in children aged less than five years worldwide and the sixth leading cause of death in seniors over age 65 in the US. Streptococcus pneumoniae represents the main etiological agent of the disease. Moreover, mortality after influenza A virus (IAV) infection has been suggested to be mainly due to secondary pneumoccocal infections. Intriguingly, death in pneumococcal-induced pneumonia can occur days after initiation of antibiotic therapy, when tissues are sterile and the pneumonia is clearing. This mortality correlates with the presence of pneumococcal virulence factors, the most important one of which is the pore-forming toxin pneumolysin (PLY). A major complication of severe pneumonia is permeability edema, characterized by a dramatically increased pulmonary endothelial hyperpermeability and an impaired alveolar liquid clearance (ALC). The enzyme PKC-(, which is activated by PLY-mediated Ca2+ influx, has recently been suggested to be implicated in the downregulation of the epithelial sodium channel (ENaC) expression, as well as in endothelial barrier dysfunction. Our preliminary data demonstrate that the lectin-like domain of TNF, mimicked by the TIP peptide, is able to blunt PLY-induced PKC-( activation, endothelial hyperpermeability and ALC dysfunction. Our overall hypothesis is that the lectin-like domain of TNF restores alveolar liquid clearance and improves barrier integrity during G+-infection- associated pneumonia, by means of blunting exotoxin-mediated PKC-( activation. We will test three specific aims to this purpose. First, we will unravel the mechanism by which the TIP peptide restores epithelial sodium channel activity and expression in PLY-treated alveolar epithelial cells. Second, we will determine at which step(s) in the cascade of events leading to PLY-induced endothelial dysfunction, the TIP peptide intervenes and what is the most upstream event affected by the peptide. Third, we will test the hypothesis that the lectin-like domain of TNF restores ALC and preserves pulmonary endothelial barrier integrity in PLY-treated mice, upon blunting PKC-( activation, making use of Triple mTNF knock-in mice, expressing a lectin-deficient mutant of TNF. The results of this research program can thus provide important information about mechanisms leading to permeability edema and dysfunctional ALC during bacterial pneumonia and can thus lead to the identification of novel therapeutic strategies. PUBLIC HEALTH RELEVANCE: One of the major complications of severe bacterial pneumonia is permeability edema, characterized by endothelial hyperpermeability and a dysfunctional alveolar liquid clearance (ALC) capacity. Our recent data from mouse models and in vitro studies using human endothelial and airway epithelial cells indicate that the TNF-derived TIP peptide, mimicking its lectin-like domain, restores ALC and endothelial barrier integrity upon treatment with the G+ bacterial exotoxin pneumolysin (PLY). This project aims to demonstrate that the protective activity of the TIP peptide in bacterial pneumonia relies on its capacity to blunt PLY-induced Protein Kinase C-( activation, which plays a crucial role in both the induction of endothelial hyperpermeability and in the down-regulation of the epithelial sodium channel, as such providing a basis for novel therapeutic strategies to combat severe bacterial pneumonia.

描述（由申请人提供）：重症肺炎是全球5岁以下儿童死亡的主要单一原因，也是美国65岁以上老年人死亡的第六大原因。肺炎链球菌是该病的主要病原体。此外，甲型流感病毒（IAV）感染后的死亡率已被认为主要是由于继发性结膜炎感染。有趣的是，肺炎球菌引起的肺炎死亡可能发生在抗生素治疗开始后几天，此时组织是无菌的，肺炎正在清除。这种死亡率与肺炎球菌毒力因子的存在相关，其中最重要的一种是成孔毒素肺炎球菌溶血素（pneumolysin，pneumolysin）。严重肺炎的主要并发症是渗透性水肿，其特征在于肺内皮细胞通透性显著增加和肺泡液体清除率（ALC）受损。PKC-β是由PLY介导的Ca ~（2+）内流激活的一种酶，最近被认为与上皮钠通道（ENaC）表达的下调以及内皮屏障功能障碍有关。我们的初步数据表明，由TIP肽模拟的TNF的凝集素样结构域能够钝化PLY诱导的PKC-β激活、内皮通透性过高和ALC功能障碍。我们的总体假设是，TNF的凝集素样结构域通过钝化外毒素介导的PKC-β激活，在G+感染相关性肺炎期间恢复肺泡液体清除并改善屏障完整性。为此，我们将测试三个具体目标。首先，我们将阐明TIP肽在PLY处理的肺泡上皮细胞中恢复上皮钠通道活性和表达的机制。其次，我们将确定在导致PLY诱导的内皮功能障碍的事件级联中的哪个步骤，TIP肽进行干预，以及什么是受肽影响的最上游事件。第三，我们将利用表达TNF的凝集素缺陷突变体的三重mTNF敲入小鼠，在PLY处理的小鼠中，在钝化PKC-β活化后，TNF的凝集素样结构域恢复ALC并保持肺内皮屏障完整性。因此，这项研究计划的结果可以提供有关细菌性肺炎期间导致渗透性水肿和ALC功能障碍的机制的重要信息，从而可以确定新的治疗策略。 公共卫生相关性：严重细菌性肺炎的主要并发症之一是渗透性水肿，其特征在于内皮细胞通透性过高和肺泡液体清除（ALC）能力功能障碍。我们最近从小鼠模型和使用人内皮和气道上皮细胞的体外研究中获得的数据表明，TNF衍生的TIP肽，模拟其凝集素样结构域，在用G+细菌外毒素肺炎球菌溶血素（pneumolysin，Pneumolysin）治疗后恢复ALC和内皮屏障完整性。该项目旨在证明TIP肽在细菌性肺炎中的保护活性依赖于其减弱PLY诱导的蛋白激酶C-β活化的能力，该蛋白激酶C-β活化在诱导内皮高通透性和下调上皮钠通道中起关键作用，因此为对抗严重细菌性肺炎的新治疗策略提供了基础。